Quantitative RT-PCR of pSV3-neo - MATERIAL AND METHODS

2 MATERIAL AND METHODS

2.9 Quantitative RT-PCR of pSV3-neo

Genomic DNA was isolated from SV40Tag cell clones C1 - C4 by a protease K digest followed by isopropanol precipitation. To detect pSV3-neo sequence we used primers, which amplify a 65 base pairs (bp) fragment corresponding to the N-terminal region of the Tag gene, SV40_1F: 5´-GATGGCATTTCTTCTGAGCAAA-3´ and SV40_1R: 5´-GAATGGGAGCAGTGGTGGAA-3´ (McNees et al., 2005). As internal reference served a region from the third exon of BDNF, a single copy gene located on rat chromosome 3, which was amplified with BDNF_F: 5´-GGACATATCCATGACCAGAAAGAAA-3´ and BDNF_R: 5´-GCAACAAACCACAACATTATCGAG-3´

(Molteni et al., 2002). Quantitative RT-PCR was performed (by Dr. Ratzka) in 96-well plates using the 7500 Fast System instrument (Applied Biosystems) running with the standard cycling program (50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15´´ and 60°C for 40´´). Finally, a dissociation curve was calculated for each well, to ensure specificity of the PCR product. The 14 µl reaction mix contained 5µl DNA (corresponding to 10 ng genomic DNA or dilution series of plasmid DNA), 3 µl primer mix (5.25 pmol of F and R primers, respectively) and 7 µl Power SYBR-Green PCR Master Mix (Applied Biosystems).

Samples were run in duplicates.

2.10 Western blotting

Harvested cell pellets (either immortalized or physiological) were homogenized with lysis buffer containing 1 % sodium dodecyl sulfate (SDS). After vortexing and sonification, the solutions were centrifuged (15 min, 15000 rpm, 4°C) and the supernatants were isolated.

Total protein concentrations were determined by biochronic acid (BCA) assay (Pierce). For detection of proteins secreted by transfected cells, 100 µl of cell condition media was collected and then centrifuged for 1-1.5h under reduced pressure (Con-1000, Con-Jet II;

Fr6bel Labortechnik, Lindau, Germany). The dry residues were reconstituted with 15 µl of water. Appropriate amounts of protein were mixed with Laemmli buffer (2x) in a 1:2 ratio, and heated at 95°C for 5 min. Equal amounts of proteins were analyzed by SDS polyacrylamide gel (12%) electrophoresis (SDS-PAGE) and western blotting onto

nitrocellulose membrane hybond ECL (Amersham biosciences) with transfer buffer (20 mM Tris, 192 mM Glycin, 20% methanol) at 120V for 1h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. C-terminus FLAG-tag, SV40Tag, GDNF, TH and GAPDH detections wereperformed after blocking the membranes (5 % milk powder in PBS containing 0.1% Tween 20 [Roth]) using the respective first antibodies (see table 2) and an enhanced chemiluminescence system (GE Healthcare).

2.11 Calcium imaging recordings, fast solution exchange

Cultured neuronal stem cells (SV40Tag clone 2) were placed in a recording chamber (3 ml), which was continuously background superfused (10 ml/min). Standard extracellular solution contained HEPES 11.6 mM, Na⁺ 129.1 mM, Cl⁻ 143.8 mM, K⁺ 5.9 mM, Mg²⁺ 1.2 mM, Ca²⁺

3.2mM and glucose 10.0 mM at pH7.3 (NaOH). Coverslips were incubated for 20 minutes in an incubator with the membrane permeable ester form of the high-affinity ratio-metric calcium dye FURA 2 AM (4μM) and allowed to de-esterify for 15–30 min at room temperature (25°C). Fluorescent images were obtained at high spatial resolution (0.09 Am2 pixel size, Till Vision Imaging System by TillPhotonics, Germany) at recording rates of 5 Hz.

For the analysis of Ca²⁺transients, background subtraction was used and subcellular regions of interest were defined over the cytosol, nucleus and neurite. Values are given as mean ± S.

E. M. To achieve fast application and removal of kainate (KA) without causing perturbation of the fluorescence signal, a custom made solution applicator was attached to the objective (Achroplan 0.75W, Zeiss, Germany) of the upright microscope (Axioskop 2 FS Plus, Germany).

The applicator were mounted directly to two canulas (0.2mm inside diameter) as described previously (Grosskreutz et al., 2007), however, the canulas were controlled by a self-constructed two-way valve-system. The perfusion rate was adjusted using a custom water-column based air pressure system fitted on 125ml reservoir syringes. The applicator was started using short 2 s pulses of 100 µM KA. Chemicals were purchased from Sigma, Germany.

2.12 Lesioning and transplantation

Adult female Sprague-Dawley rats with 220-240 g weight received two stereotactic unilateral 6-OHDA (Sigma-Aldrich) injections in the medial forebrain bundle (MFB) under ketamine/rompun anesthesia [per animal: 0.25 ml ketamine hydrochloride (5%; 1 ml = 57.67 mg, 0.1 ml/100 g) plus 0.05 ml xylazine (Rompun; 1 ml = 23.32 mg), intraperitoneally] as previously described (Timmer et al., 2004). Transplantation was performed at least 6 weeks after lesioning. The SV40Tag cells were transplanted after culturing for i) 12 days under differentiation media with or without dbcAMP/GDNF supplement respectively, and ii) 3 days in proliferation media following 12 days in differentiation with dbcAMP/GDNF supplement.

SV40Tag cells were cultured 1 day in adhesion media and 4 days in differentiation before transplantation. The cells were deattached, resuspended with 0.05% DNase/DMEM and adjusted to a final density of 100.000 cells/µl. The stereotactic intrastrial transplantation was perfomed using a glass capillary attached to a 2µl Hamilton syringe as described by Nikkhah et al. (Nikkhah et al., 1994b), using the stereotactic coordinates as described by Timmer et al. (Timmer et al., 2004). Each rat received 4 deposits of the suspension 1 µl each, into the right lesioned striatum. Seven and 14 days after transplantation, the rats were perfused with 4% paraformaldehyde (PFA). The brains were quickly dissected, afterwards postfixed in 4%

PFA overnight and passed into 30% sucrose in PBS. Coronal sections were cut on a freezing microtome at 30 µm thickness. Cryoprotection was done at -80^oC in anti-freeze-medium (30% glycerine (v/v), 30% ethylenglycol (v/v), 40% PBS).

2.13 Immunocytochemistry

The immunocytochemical staining was performed according to Timmer et al. (Timmer et al., 2006). Cells were fixed with 4% PFA in PBS for 20 min at RT followed by three washing steps with PBS. Afterwards, cells were incubated in blocking buffer (PBS containing 0,3% Triton X-100 and 3% normal goat serum (NGS)) for 60 min at RT. The primary antibodies were diluted in blocking solution and incubated overnight at 4°C. Then, following three washing steps with PBS, the secondary antibodies were applied to the cultures for 1 hour (in the dark). For detection of bound primary antibodies IgG Alexa Fluor 555 and 488 conjugated secondary antibodies (see table 2) were applied. Nuclei were visualized by

4,6-diamidino-2-30

phenylindole(DAPI, Sigma-Aldrich) staining when required.Cells were washed again (three times with PBS) and then were directly analyzed in the plates using an Olympus fluorescent microscope.

2.14 Immunohistochemistry

Different time points after transplantation surgery, animals were deeply anaesthetized with ketamine and rompune and perfused transcardially with 50 ml of 0.9% saline followed by 250 ml of 4% PFA in PBS. The brains were post-fixed overnight and immersed in 3% sucrose.

Serial coronal sections were cut on a freezing microtome at 30 μm thickness (3 series). In order to detect DA neurons every sixth section was processed for TH immunohistochemistry in free-floating manner, using the avidine-biotin-complex (ABC) kit and 3´,3-diaminobenzidine (DAB) for visualization. The blocking of endogenous peroxidase activity and cell permeabilization was performed by incubating the slices with 3% H2O2 / 10%

Methanol solution in PBS for 10 min in RT. Following three times washing primary antibody (monoclonal mouse anti-TH, 1:200, Chemicon) diluted in blocking buffer was applied overnight at 4°C. From this point on every step was followed by subsequent three times washing with PBS. Then the slices were incubated with biotynilated secondary antibody (anti-mouse, 1:200) diluted blocking buffer (containing 1% BSA, 0,3% Triton-X in PBS)for 1 hour at RT. Freshly prepared ABC (incubation of 30 min before application at RT) was applied to the slices for 1 hour at RT. Finally, the staining reaction was developed with 0.05% DAB / 0.02% H2O2 and ammonium-nickel sulfate, in the dark. The slices were mounted with Corbit Balsam and analyzed under the microscope. Transfected (with EGFP) VMP cells were detected by fluorescence microscopy. For SV40Tag immunohistochemical staining, biotinylated polyclonal rabbit anti-mouse immunoglobulins (Dako), Vectastain Avidin-Biotin-Complex (ABC) Kit (Linaris), mouse IgG and 3´, 3-diaminobenzidin (DAB) (Sigma-Aldrich) were used.

2.15 Bioassay neurotrophic factors for DA neurons

2.15.1 Small scale expression of neurotrophic factors for DA neurons

For small scale expression of a set of NTFs and transcription factors, freshly prepared dissociated VM neuronal progenitor cells isolated from E12 cells, were expanded in 6well-plates with a density of 600.000-800.000 cells/well, 1 day in 2 ml of adhesion media followed for 2 days in proliferation media. At DIV3 cells were detached, counted and seeded in 96well-plates in a density of 40.000 cells/well in 200 µl of adhesion media. At DIV7 transfection using Lipofectamin 2000 was performed as previously described, for overexpression of a set of candidate factors to induce differentiation, maturation, and/or neuroprotection in DA neurons. The following day, medium was changed to differentiation media. At DIV10 condition mediums were collected, and after 7 days in differentiation conditions (DIV14) cells were fixed with 4% PFA in PBS for 15 min, and then washed twice with PBS. Further, ICC was performed to quantify the effects of NTFs on the number of DA (TH-positive cells) neurons, or together with nuclei visualization by DAPI, estimate transfection efficiency (ratio FLAG-M2 positive / DAPI positive).

2.15.2 Imaging methods and counting

The TH-immunoreactive (TH-ir) neurons were analyzed by fluorescence microscopy (Olympus IX70) and software AnalySIS Pro (version 3.2, www.soft-iamging.de). Photos were taken in a 4x magnification of 5 random fields of the well, representing 1/3 of the total area of the well (96-well plates). To avoid regions without homogeneous density of cells, the fluorescence channel showing the DAPI stained nuclei was first used. Next the fluorescence channel showing the anti-TH staining was used to take the photographs. Analyze of taken photos was done using the program ImageJ (version 1.41, http://rsb.info.nih.gov/ij).

Neurons were counted manually on each photo by tagging the neuron. Different object classes could be counted per image. Therefore, it was possible to count TH-ir cells were the cell body was clearly noticeable. These cells were then categorized with regard to the number of primary neurites sprouting from the soma of cells; e.g. cells with two neurites (these cells are bipolar and immature with thin cell bodies); cells with three neurites (these cells are maturing and have bigger cell bodies); and cells with at least four neurites (mature

neurons, comparatively big cell bodies classified by their number of neurites). Data were logged automatically and transferred to a spreadsheet (Excel). The ImageJ software supports automatic counting. However, it was not possible to resolve individual neurons when they are in clusters. Moreover, was not possible to set the filters for the acquisition parameters to avoid the inclusion of debris, and therefore gross errors can result. Manual counting seems more accurate. In addition, double-counting can be avoided completely, since each cell is marked, and different object classes can be marked with different colors. To verify the expression of the transfected factors, cells were stained against FLAG, which could be co-related with the protein expression in cell lysates and respective condition mediums, collected after the same culture conditions.

2.15.3 Neuroprotective assays

To evaluate neuroprotective effects of different factors on DA neurons, 2 different assays were tested, including i) exposure to the neurotoxin 6-OHDA and ii) serum deprivation.

i) Administration of 6-OHDA, a neurotoxin for DA neurons

To evaluate putative neuroprotective effects on DA neurons by expression of NTFs, 6-OHDA-treatment was performed in cultures after six days under differentiation conditions (as described 2.15.1), after which an additional medium change containing 40, 70 or 100 µM 6-OHDA (stock solution: 10mM 6-6-OHDA in DMEM) was added to the cultures. Control groups received 6-OHDA was replaced by PBS. Cells were then fixed and processed for immunocytochemical investigations after 16, 18 or 21 hours after exposure to the toxin (Grothe et al., 2000; Yuan et al, 2008).

ii) Serum deprivation

Complete serum deprivation has been reported to cause relatively rapid death of DA neurons (Takeshima et al., 1994) depending on the growth medium used. Based on this effect, we cultured genetically modified VM neuronal progenitor cells as previously described (in 2.15.1) however, with modification of the media composition, in means of FCS absence. In control groups cell cultures were exposed to the standard differentiation media (including 1% FCS serum). After 7 days under these conditions, cells were then fixed and processed for immunocytochemical investigations.

2.16 Statistical analysis

Results are expressed as means ± standard deviation. Statistical evaluation was performed using GraphPad InStat program (version 3.06). Comparison between the groups were performed by using one-way analysis of variance (ANOVA) followed by Tukey posthoc test if the data was normally distributed or by Kruskal-Wallis one way analysis of variance on ranks followed by Dunn´s posthoc test if the data was not normally distributed.

3 RESULTS

3.1 Characterization and differentiation potential of SV40 large T antigen-immortalized rat VM neuronal progenitor cells

3.1.1 Generation of immortalized VM neuronal progenitor cell clones

Ventral mesencephalic progenitor cells, prepared from E12 rat embryos, were cultured according to Timmer et al. (Timmer et al., 2006). After 3 days under proliferation conditions, cells were transfected non-virally by nucleofection to express SV40Tag. Following two weeks under selection media, clones were obtained by limiting dilution. Presence and expression of SV40Tag was monitored in four cultured neuronal progenitor cell clones (C1, C2, C3 and C4).

Semiquantitative RT-PCR revealed expression of SV40Tag mRNA in the different cell clones (Fig. 5A); western blot analysis showed the presence of the SV40Tag protein in the cell clones (Fig. 5B). Immunocytochemical staining for SV40Tag was found in cultures of immortalized cell clones in vitro (Fig. 5c-f) and after transplantation in the parkinsonian rat model (Fig. 10A-B). Moreover, cellular DNA isolated from the four SV40Tag cell clones (C1 – C4) revealed beside the high molecular genomic DNA two additional bands at 750 and 1500 base pairs, which were not present in a control genomic DNA preparation isolated from an adult rat (data not shown). As the size of those bands was much smaller compared to the supercoiled pSV3-neo plasmid (8.5 kb) also loaded on the gel, those bands most likely represent recombined plasmids. Such extra-chromosomally replicated DNA has been already reported for cells transfected with the ori containing pSV3-neo plasmid (Ray et al., 1990;

Legrand et al., 1991). Normalization of the cellular DNA amounts with the single copy gene BDNF by quantitative PCR, revealed similar amounts of SV40Tag sequences in line C1, C2 and C4, whereas line C3 contained only half the amount (data not shown).

Figure 5: Expression of SV40Tag in SV40Tag-immortalized cells. (A) Semi-quantitative RT- PCR shows the mRNA expression of SV40Tag. (B) Western blot analysis shows the presence of the SV40 large Tag (90kDa) and SV40 small tag (19Kda) proteins in different cell clones. Primary cells (E12) were used as negative control. Immunocytochemistry showing SV40 (inserts C’-F’) and Nestin (C-F) staining of immortalized cells in vitro. Nuclei were visualised by DAPI. [Clone 1 (C1), clone 2 (C2), clone 3 (C3) and clone 4 (C4)]. Scale bar = 50 μm.

3.1.2 Reduced doubling time of SV40Tag clones

To study the proliferation activity, doubling time was evaluated using the WST-1 assay. The growth behaviour of the immortalized cell clones was compared to the growth of primary mesencephalic progenitor cells. All cell clones analyzed displayed a two-to three-fold lower doubling time as compared to the naive E12 cells (Fig. 6A). Doubling time of the immortalized cell clones ranged between 13 to 18 hours while for primary E12 cells it was 47 hours (Fig. 6A). Furthermore, the selected cell clones were passaged for up to 25 times, corresponding to more than 120 doubling times, and remained responsive to FGF2, with no apparent changes in growth rate.

3.1.3 Expression of markers for specification and early differentiation of DA neurons Characterization was performed with regard to a molecular profile of transcription factors and other molecules related to DA differentiation (Table 1) using semiquantitative RT-PCR and morphological analysis. Primary mesencephalic cultures and immortalized cell clones were analyzed under proliferating and differentiating conditions (Timmer et al., 2006). First, characteristic profiles for primary mesencephalic progenitor cells were observed after 2 and 4 days under proliferation, followed by additional 2 and 5 days under differentiation conditions, respectively (Fig. 6B). The following genes were analysed: paired box 2 (Pax2), LIM homebox transcription factor 1 (Lmx1b), wingless-type MMTV integration site 1 (Wnt1), wingless-type MMTV integration site 5 (Wnt5), nuclear receptor subfamily 4, A2 (Nurr1), delta-like 1 homolog (Dlk1), engrailed factor 1 (En1), paired-like homeodomain 3 (Pitx3), neurogenin 2 (Ngn2), dopamine transporter (DAT) and tyrosine hydroxylase (TH). Under differentiation conditions an increased expression level of terminal markers for DA neurons such as Pitx3, DAT and TH can be seen. Furthermore, we analysed the immortalized mesencephalic cell clones after short culture duration (1 day in adhesion media plus 3 days differentiation). Concerning genes involved in specification and early differentiation towards DA neurons C2, C3 and C4 revealed a similar profile as compared to the primary VM cultures (Fig. 6C), such as the expression of Lmx1b (lower in C3), Wnt1, Wnt5, Nurr1, En1 and Dlk1.

However, neither expression of Ngn2 nor any of the terminal differentiation markers such as Pitx3, TH and DAT were detected (data not shown). Under these conditions, cells remained morphologically undifferentiated, which was supported by ICC, were neither neuronal

(β-37

tubulin type III) nor glial cells (glial fibrillary acidic protein (GFAP)) were found (data not shown), whereas all the cells were SV40 (Fig. 5c-f) and nestin immunoreactive (Fig. 5C-F).

Figure 6: Characterisation of generated SV40Tag-immortalized cell clones. (A) Growth behaviour of immortalized cell clones in comparison with primary mesencephalic progenitor cells. Using WST-1 assay, the actual reagent absorbance of the different cell clones was measured at two different time points, with an interval of 24h, and doubling times of the generated cell clones and primary NPCs were calculated (C1 n=5, C2 n=3, C3 n=3, C4 n=5, and E12 n=3). Values represent mean ± SD doubling times in hours from n experiments. (B,C) Expression levels of characteristic DA neuronal markers during different phases of differentiation towards DA phenotype in vitro. (B) E12 ventral mesencephalic NPCs express mRNA for TH, DAT, Pitx3, En1, Wnt1, Wnt5a, Dlk1, Pax2, Nurr1, Lmx1b and Ngn2. The expression of Pitx3, DAT and TH significantly increases under differentiation conditions. (C) SV40Tag-Immortalized cells clones after 3 days under differentiation conditions, displaying differential mRNA expression between the different clones of Pax2, Lmx1b, Wnt1, Wnt5a and Dlk1. Whereas Nurr1, En1 are expressed in all clones. β-actin gene was used as an internal standard of input cDNA.

3.1.4 Differentiation potential of VM immortalized cell clones 3.1.4.1 Silencing of SV40 large T antigen

Cell clones were further studied with regard to their differentiation potentials using two different conditions. Expression of SV40Tag has been shown to inhibit differentiation (Truckenmiller et al., 1998); therefore, our first attempt to induce further differentiation of the selected clones was the introduction of a vector encoding SV40Tag shRNA to achieve the silencing of SV40Tag expression. Only one cell clone (C1) revealed successful silencing, shown by western blotting and ICC for SV40Tag (Fig. 7). Culturing in the presence of differentiation mediums, suitable for primary progenitor cells (Timmer et al., 2006), failed to trigger DA differentiation (i.e. TH-immunoreactivity, data not shown). However, neuronal and glial differentiation occurred under this condition, since β-tubulin type III and GFAP immunoreactive cells, respectively, were found (Fig. 8).

3.1.4.2 Application of dibutyryl cyclic AMP and GDNF

The second differentiation approach was the culture of immortalized cell clones (C2 and C3) under differentiation conditions in the presence of dbcAMP and GDNF for 12 days.

Interestingly, evaluation of the expression of different genes involved in DA neurogenesis, revealed one cell clone (C2) displayed an expression pattern similar to DA neurons present in culture of primary NPCs, including expression of Pitx3, DAT and TH (Fig. 9I). Additional differences were observed when the expression profile of C2 and C3 after 3 days (Fig. 9B) was compared with 12 days of differentiation (Fig. 9I). Expression of Pax2, which was detected in C3 after 3 days differentiation, is vanished after 12 days differentiation. In contrast, expression of Ngn2 was not detected in any cell clone after 3 days, however was observed in both C2 and C3 clones after 12 days differentiation. Differences in morphology between dbcAMP/GDNF treated and non treated cells, in particular number and length of sprouting neurites were observed after nestin staining and phase contrast analysis. Further neuronal differentiation (marked by β-tubulin type III immunoreactivity) was found in the presence of dbcAMP and GDNF treatment for 12 days (Fig. 9A-H).

Figure 7: Transfection with the plasmid pSUPER.puro encoding shRNA for SV40Tag induced suppression of SV40 immunoreactivity. (A) Western blot showing downregulation of SV40Tag expression 6 days after transfection with pSUPER.puro.SV40Tag, and as control, cells transfected with the empty pSUPER.puro were used. Cultures were under puromycin (1mg/ml) treatment for selection of transfected cells. GAPDH was used as loading control. Immunocytochemistry revealed a clear decrease in SV40Tag immunoreactivity in the majority of cells transfected with pSUPER.puro.SV40Tag (C, G). Control cells, transfected with the empty pSUPER.puro were SV40Tag immunopositive (B, F). Both groups of cells were treated with puromycin for 6 days after transfection. DAPI nuclear stain of the respective field (D-G). Scale bar = 50 µm.

Figure 8: Neuronal and glial differentiation after transfection with the plasmid encoding SV40Tag shRNA. Increased number of β-tubulin type III immunoreactive cells was observed after 4, 6, 8 and 12 days after silencing (A, B, C and F respectively). A low number of GFAP immunopositive cells were observed after 12 days (E, H). In addition, cells remained nestin positive after 12 days (D, G).

Nuclei visualisation by DAPI (G, H and I). Scale bar = 50 µm.

3.1.5 Expression of kainate-regulated calcium permeable glutamate receptors

Fura-2 fluorescent calcium imaging was used to detect calcium transients in C2 cells (Fig. 9J).

The method was used to derive functional information on neuronal differentiation and its time course. In a first step recording with agonist-free extracellular solution was performed to detect spontaneous transients of intracellular calcium concentration as a functional correlation of the expression of calcium permeable synaptic glutamate receptors, i.e. a marker of neuronal differentiation, and to trace the establishment of synaptic contacts. In

Im Dokument Genetically modified ventral mesencephalic neuronal progenitor cells (Seite 27-0)